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The correlation between the chemical stability and the microbiological activity of polymyxin B in phosphate buffer pH 6.0 and in proprietary eye drops was evaluated. High Performance Liquid Chromatography (HPLC) was used to quantify the amount of the main components in samples stored at 43,50,55 and 60°C for a period of 500 h. The data indicated that there are significant differences in chemical stability among the different proprietary eye drops. The accurate decomposition rate constants and shelf-lives (~o) at 4°C of two of the six formulated eye drops and the standard polymyxin B solution stored in glass containers at pH 6.0 were established. It was concluded that microbiological assay by agar diffusion was unsuitable for determining the activity of control polymyxin B in phosphate buffer and polymyxin B in eye drops. Killing time determinations for polymyxin B against cell suspensions of P. aeruginosa NCTC 6750 were consequently used. Thioglycollate broth containingp- aminobenzoic acid (PABA) 0.16 %w/v and magnesium sulphate 1 %w/v was used as an inactivating recovery medium. The effect of preservatives and of second antibacterials contained in the eye drops were tested individually and combined with polymyxin B. Thiomersal 0.001 %w/v, trimethoprim 0.02 %w/v and thiomersal 0.001 %w/v plus trimethoprim 0.02 %w/v did not have an effect on the activity of polymyxin B 2000 U/ml. Neomycin was an exception and at the concentrations in the range 0.0192 to 0.16 %w/v exhibited an antagonistic effect. Chemical interaction between polymyxin B and neomycin could not be detected and it was considered that the inhibitory effect of neomycin may be the result of competition between polymyxin B and neomycin for the same binding sites on the cell surface. Gentamicin is active against P. aeruginosa NCTC 6750 and at concentrations of 0.075 and 0.036 %w/v it exhibited an additive effect with polymyxin B 2000 U/ml against the test organism. The results obtained with the samples stored at 55°C for a period of 500 h demonstrated the critical effect of pH. At a pH of 6.0 microbiological activity and chemical stability appeared optimal. The chemical stability data of five eye drop samples correlated with microbiological activity data. Exceptions were polymyxin B in one eye drop sample and control polymyxin B. These extensively decomposed samples showed good antibacterial activity which appeared to result from the activity of decomposition products. Chemical stability data for standard polymyxin B solution at pH 6.0 also correlated to microbiological activity data over the temperature range of 92 - 115°C. The polymyxin B retained detectable microbiological activity when the amount of PB1 was greater than 20%. It is suggested that the decomposition products which occurred at these higher temperatures did not possess antibacterial activity.

I have investigated the evolutionary response of Pseudomonas aeruginosa to salt (NaCl) stress, and the physiological mechanisms responsible for this adaptation. Populations of P. aeruginosa founded from the same ancestral genotype were selected at three different concentrations of NaCl, low, moderate and high for about 660 generations with four independent replicates for each concentration. Adaptation was measured as the fitness of the evolved populations relative to the ancestor assessed in direct, head-to-head competition experiments conducted in the same environment in which they were selected (direct response) as well as in all alternative environments (correlated response). Results suggest that selection in each salt environment led to adaptation to that environment and a modest degree of specialization that evolved because correlated responses to selection were smaller than direct responses. In order to identify the physiological mechanisms contributing to the populations' adaptation in high NaCl concentration, I chose a sample of evolved lines that showed the strongest evidence for specialization to salt and competed them against the common ancestor in KCl and sucrose. Results suggested that increased Na+ /H+ antiporter activity is probably the primary mechanism behind adaptation to high NaCl concentration, however alternative mechanisms cannot be excluded. Tolerance curves, which measure the performance of a genotype across a gradient of salt concentrations, suggested no change in the high salt group’s ability to tolerate extreme concentrations of NaCl. We conclude that high salt evolved population showed improvements to its ionic/osmotic stress resistance strategies mainly to Na+ efflux strategies but with no changes to salt niche.

Cystic Fibrosis is one of the most common genetic recessive diseases among Caucasians and is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene on chromosome 7. There are different classes of CFTR mutation, leading to differences in disease severity among patients. In addition to the CFTR genotype, secondary genetic factors, modifier genes, also influence CF phenotypes. Due to the dysfunction of CFTR protein and production of thickened mucus, bacterial infection in the lungs is favored and can lead to further clinical complications in CF patients. Pseudomonas aeruginosa is one of the most common bacteria detected among patients. The aim of this project was to investigate four candidate modifier genes, Factor B, Complement Factor 3, Toll-like Receptor 4 and Heme oxygenase-1, which might affect the status of Pseudomonas aeruginosa infection. A total of 22 single nucleotide polymorphisms (SNPs) were selected in these four genes and they were tested against five phenotypic traits, including age of diagnosis, FEV1% predicted andstandard deviation value, age of first Pseudomonas aeruginosa infection and Pseudomonas aeruginosa infection status. Among the selected SNPs, both case-control studies and family-based analysis were performed in order to establish any correlation between the genotypes and the phenotypes. In addition, haplotype analysis was performed to determine whether there was interaction between SNPs or whether there were unidentified SNPs in the vicinity of the selected ones that might contribute to the observed phenotypic traits. Among the 22 chosen SNPs, 13 of them were found to be significantly linked to one or more of the tested phenotypes. The three most significant associations were BF_2557 with lung function, HMOX1_9531 with lung function and BF_7202 with age of diagnosis. Several haplotypes were significantly associated with one of the five phenotypes. There was no evidence for the presence of unidentified SNPs or interaction between SNPs. Most of haplotype associations were likely due to the presence of a single SNP which was found to be significantly linked to the phenotype. Conclusively, both SNPs and haplotype analyses suggest that the four candidate genes are modifiers of disease severity in CF.

Doctor of Philosophy / Cystic fibrosis (CF) is the most common inherited lethal disorder among Caucasian populations. Chronic pulmonary infections, particularly from Pseudomonas aeruginosa, are the major determinant of the morbidity and mortality of people with CF. It is generally accepted that people with CF acquire this pathogen independently from their surrounding environment, and that individual CF patients carry unique strains different from others. The spread of this pathogen from patient to patient is thought to be rare and occurs particularly among closely contacted cases such as CF siblings. However, over the past decade, there have been several reports of an emergence of clonal P. aeruginosa strains commonly found infecting a number of CF patients. One such report is from the CF paediatric clinic at the Royal Children’s Hospital in Melbourne in which more than half of the patients were infected with a single strain or clone, subsequently called Australian epidemic strain 1 or AES-1. A preliminary survey showed that AES-1 had spread extensively along the Australian eastern seaboard among CF patients attending other CF centres in Melbourne, Sydney and Brisbane, including adult patients at the Royal Prince Alfred Hospital (RPAH), Sydney. Another clonal strain, subsequently called AES-2, was identified in both CF adults and children at the Prince Charles Hospital and the Royal Children’s Hospital, in Brisbane. The total extent of prevalence of the AES-1 and AES-2 strains at the RPAH as well as the clinical status of patients who carried these strains was unknown. Moreover, the pathogenicity of these two clonal strains had not been investigated. The studies presented in this thesis investigated the prevalence of these clonal strains among CF patients attending the adult CF clinic at RPAH, Sydney by using pulsed-field gel electrophoresis. Overall, 50% of 112 patients with P. aeruginosa were found to be infected with clonal strains. The AES-1 and AES-2 strains were identified in 38% and 5% of the patients respectively. Two new clonal strains, called Sydney-1 and Sydney-2, were also identified. Patients with clonal strains had a significant increase in their number of exacerbations and hospitalisation days, and tended to have lower pulmonary functions when compared to patients infected with non-clonal strains. By using a variety of bioassays to examine the pathogenicity of the clonal and non-clonal strains, it was found that both AES-1 and AES-2 produced more virulence factors and were more resistant to antibiotics when compared to the non-clonal strains. AES-1 and AES-2 were associated with increased production of proteases, including elastase, alkaline protease and protease IV. Overall the results presented in this thesis suggest that there may be a link between virulence and transmissibility of this pathogen. The studies presented in this thesis also compared the biofilm forming capacities of the AES-1 and non-clonal isolates. AES-1 was shown to have greater biofilm-forming capacity than the non-clonal strains, when they were grown on a glass surface, suggesting a possible association between clonality and biofilm formation. A model for the study of bacteria grown in conditions similar to CF sputum was also developed. P. aeruginosa grown in this model was found to develop into clumps which may be comparable to the biofilm structure in the CF lung. This model was shown to be beneficial for transcriptomic and proteomic studies which are underway within the research group. AES-1 was also found to have phenotypic variations between isolates. By applying the amplified fragment length polymorphism technique, more subtypes of this clone were revealed. However, these detected subtypes did not correlate with the different phenotypes, suggesting minor mutations such as single point polymorphisms may be responsible for the phenotypic diversity within the clone. The final part of this thesis was devoted to examining the safety of a novel CF treatment: hypertonic saline (HS) inhalation. HS was shown to increase airway mucociliary clearance, while increased osmolarity associated with the use of HS was also shown to have an inhibitory effect on the formation of biofilms. Findings in this study proved that there was no evidence of strain selection in patients who received the long-term treatment with HS. The study also demonstrated that AES-1 was significantly more persistent in the CF lung than the non-clonal strains. The present thesis not only defines the clonal strains of P. aeruginosa and their implications for infected patients, but also provides a general understanding into the pathogenesis of both clonal and non-clonal strains infecting CF lungs.

Thesis (PhD Microbiology and Plant Pathology(Water resource management))-University of Pretoria, 2006. / Summary in English. Includes bibliographical references. Available on the Internet via the World Wide Web.

Professional paper (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Michael Franklin. Includes bibliographical references (leaves 37-46).

Thesis (Ph.D.)--Ohio State University, 1977. / Includes vita. Includes bibliographical references (leaves 127-140). Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [67]-74).

Diss. Naturwissenschaften Zürich, 1990. / Rückentitel: Resistenz von P. aeruginosa gegen Betalaktam-Antibiotika. Name auch: U. Vurma-Rapp.